In the present work, we report for the first time the presence of hereditary bisalbuminemia in two groups of related bottlenose dolphins identified by means of capillary zone electrophoresis and we confirm that agarose gel electrophoresis could fail in the identification of this alteration, as already reported in human medicine [7, 8].
Serum protein electrophoresis is the most reliable method to determine the distribution of serum protein fractions and is considered, together with a basic hematological and biochemical profile, an essential step to evaluate the health status of animals, providing clinically useful information. The interpretation of kinetics of total proteins and albumin and globulin fractions is receiving increased attention also in marine mammals in which, as in terrestrial mammals, a typical pathologic pattern could be identified in several diseases, such as inflammatory diseases [15]. Nowadays, in many veterinary laboratories, CZE has replaced classical agarose gel electrophoresis, due to its higher resolution. The difference in resolution is mainly due to the different analytic method: while, in AGE, proteins migrate toward anode in a solid phase and in an alkaline buffer with low voltage, in CZE proteins rapidly move in a liquid phase toward the cathode thanks to the high voltage applied. This allows a better separation of proteins with similar physicochemical characteristics, thus generating multiple sub-peaks or narrower peaks [16]. When CZE was introduced routinely in human medicine laboratories, an increased number of bisalbuminemia cases was detected [7], based on the improved separation of the albumin, α1-globulin, and α2-globulin fractions. In our work, this technique clearly identified a double albumin peak at the visual analysis of the electrophoretic profile in 9 samples whereas with AGE only 8 profiles revealed a wider peak compared to the normal ones, but never an albumin double peak was detected. Furthermore, with AGE, an albumin peak apparently wider than normal was noted also in one sample with normal CZE profile, demonstrating as the visual interpretation of AGE profiles could lead to both false negative and false positive detection of bisalbuminemia. However, the visual identification of a “wider” peak could be considered a subjective method. Thus, we calculate the ratio between the length of the base and the height of the albumin peak with the aims to define a cut-off and to established a more accurate and objective method to identify bisalbumenimia in AGE electrophoretic profile. However, also with this approach the diagnostic accuracy was fair with a low specificity.
As expected, no significant differences in TP concentration, albumin percentage and absolute values between affected and normal dolphins was noted, but CZE albumin were significantly lower compared to AGE. In literature, an opposite situation is reported with higher albumin values obtained with CZE in dogs and cats [14]. Nevertheless, it’s possible that these data, despite the significant differences, are not clinically relevant. In literature, reference ranges for TP and serum protein fractions are available for free ranging bottlenose dolphins [17, 18]: compared to our results obtained with both methods, in free-ranging dolphins TP seemed higher and albumin absolute values lower, suggesting a higher concentration of globulins in these animals, thus a tendency to an inflammatory status, as already suggested [15]. All these data highlight the need to define appropriate reference ranges for different electrophoretic methods for bottlenose dolphins under human care.
The application of higher resolution techniques, such as CZE, can result in an increased number of “abnormal” profiles and thus a deeper knowledge of the clinical importance of these new profiles is required. In previous works on CZE validation in companion animals, an unusual albumin peak was observed in sera from clinically healthy cats never been reported previously, likely because of the low resolution of traditional electrophoretic techniques [14]. Thus, the correct interpretation of these kinds of data is essential to differentiate normal to pathological conditions. In human medicine, the diagnostic implications of the presence of bisalbuminemia in clinical entities are uncertain: it could be a sign of acquired disorders and it is correlated with several pathological conditions, like pancreatic and hepatic diseases (pancreatitis, pancreatic pseudocysts, hepatic chirrosis), lymphoproliferative diseases (monoclonal gammopathy, multiple myeloma) [2]. In our case, all the sampled dolphins were clinically healthy, without any other alteration in hematological or biochemical parameters and bisalbuminemia was detected accidentally, during the routine evaluation of the health status. Furthermore, no drugs were administered to the affected animals, except for an integration of folic acid in two of them, thus we could exclude the possibility that the abnormal electrophoretic pattern identified is due to a pathological condition or drugs administration.
The inherited form of human bisalbuminemia is usually discovered by chance and apparently does not seem associated with pathological conditions. Genetically, bisalbuminemia is due to a mutation in the albumin gene transmitted as an autosomal codominant trait and it has been reported in various human populations around the world, with significant differences in frequency in terms of race and location, with higher incidence in small, isolated population groups [3]. All the dolphins sampled in our study were living under human care and, from a reproductive point of view, they include wild founders and their progeny of first and second generation maintained in four groups separated in different facilities; for this reason the high incidence of this inherited disorder is somehow concentrated and not surprising.
Since the protein synthesis is governed by a single copy gene codominantly expressed, heterozygous subjects carrying point mutations usually show the presence of the normal and the variant proteins. Based on the pedigree of affected dolphins, we could only suppose the same inheritance pattern in bottlenose dolphins, but the molecular analysis of the albumin gene in affected dolphins and their related normal animals should be carried out to investigate the genetic defect underlying and the inheritance mode of transmission.
Over the last three decades, more than 60 different albumin variants have been characterized in people, being the vast majority reflecting single-base changes in the structural gene mainly with mutations in hypermutable CpG dinucleotides [1]. Rarely, the presence of bisalbuminemia may have a clinical impact due to the effect of mutation on ligand-binding: three mutations (p.Leu90Pro; p.Arg242His; and p.Arg242Pro) form strong binding sites for triiodothyronine (T3) or thyroxine (T4), causing the familiar dysalbuminemic hypertriiodothyroninemia, and the familiar dysalbuminemic hyperthyroxinemia syndromes [19, 20]. Other mutations seem to increase the binding capacity of long-chain fatty acids, but without clinical consequences [21]. Apparently, no signs of altered hormone or lipid binding capacity were evident in sampled dolphins based on the absence of laboratory abnormalities, but molecular information on genetic variants and mutations are needed to obtained valuable data about albumins binding properties.